Effect of cocaine on NO levels, and NOS inhibition on MOR protein and mRNA levels. (A) Representative images depicting DAF-2 fluorescence in (i) control (untreated) cells and cells treated with (ii) 500 μM SCT, or (iii) 100 μM RIT with cocaine for 72 h. The left column depicts fluorescence, the middle column is the corresponding Nomarski differential interference contrast image, and the right column is the overlay of these two images. Basal fluorescence was detected in control treated PC12 cells. Following exposure of cells to 500 μM SCT and 100 μM RIT, fluorescence intensity increased. The cocaine-induced increase in DAF-2 fluorescence was prevented when cells were pretreated with 20 mM L-NAME in both treatment regimens (iv and v). (B) Representative immunoblot of MOR protein (upper panel) and α-tubulin (lower panel) levels obtained from lysates of control (untreated) and cocaine-treated PC12 cells grown in the presence or absence of 20 mM L-NAME. Cells were exposed to 500 μM SCT or 100 μM RIT with cocaine for 72 h. (C) Densitometric analysis of MOR protein expression relative to α-tubulin in control, cocaine and L-NAME treated cells. Relative to control, cocaine (SCT and RIT) significantly increased relative MOR protein levels and this increase was prevented in cells pretreated with L-NAME. (D) qPCR analysis of MOR mRNA expression relative to β-2 microglobulin in control, cocaine (100 μM RIT) and L-NAME (alone or in combination with 100 μM RIT cocaine) treated PC12 cells. Relative to control, 100 μM RIT of cocaine significantly increased MOR mRNA, and this increase was prevented by pre-treatement with 20 mM L-NAME. Results are representative of at least 5 independent experiments and the data are shown as mean ± SEM (*p<0.05, ***p<0.001).